1. Field of the Invention
The present invention relates to multizone analytical elements which are useful for the determination of an analyte in a liquid test medium. In particular, the present invention relates to multilayer immunoassay test devices involving the use of labeled reagents comprising a chemical group having a detectable physical property such as fluorescence or color.
2. Description of the Prior Art
Multizone analytical elements or test devices have been previously proposed and have been applied to binding assays, e.g., immunoassays, which depend upon the ability of an antibody or antigen to bind to a specific analyte for the determination of the analyte in the liquid test medium. Such assays include those immunoassays where a labeled reagent, such as a labeled form of the analyte or an antibody thereto, participates in an antigen-antibody reaction to form a free species and a bound species thereof such that the amount of the labeled reagent in one of such species can be correlated to the amount of analyte in the liquid test medium. In principle, such assays are referred to as heterogeneous immunoassays because the free and bound species must be separated in order to complete the assay.
Multizone, particularly multilayer, analytical elements are now known in the art which inherently perform the required separation step so that no additional manipulations are needed after application of the liquid test medium. In general, such devices include a plurality of layers having the necessary reagents for carrying out an immunoassay and for accomplishing the necessary separation step incorporated therein. A number of such devices further include a detection layer from which the signal produced by a labeled reagent in either the bound or free species is detected and measured. Detectable signals provided by such devices are usually optical in nature such as color changes, fluorescence, or the like. Alternatively, detection can be accomplished by electrochemical measurements using, for example, potentiometric or ampometric techniques.
For example, such multilayer immunoassay analytical elements are described by European Patent Publication No. 97,952 and German Publication No. DE-OS No. 3329728 where an immobilized form of a binding partner, such as an immobilized antibody to an antigen, and an antigen labeled with a detectable substance are incorporated therein. Upon the application of a liquid test medium to such device, antigen from the test medium competes with labeled antigen incorporated into the device for binding to the immobilized antibody. Separation of the bound species from the free species occurs upon migration of the free species of the labeled antigen away from the immobilized zone.
Similarly, European Patent Publication Nos. 51,183 and 66,648 disclose such devices where the determination of antigen or antibody in a liquid test medium is dependent upon the competitive binding of the antigen (or antibody) with a labeled form of the antigen (or antibody) for an immobilized form of a binding partner thereof, such as immobilized antibody (or antigen).
Other multilayer immunoassay test devices have also been proposed, such as described in U.S. Pat. No. 4,258,001, which include one or more layers comprising particulate, three-dimensional lattices formed by a plurality of organopolymeric particles. The particles form interconnected void spaces which are claimed to provide for the transport of high molecular weight analytes therethrough. Although not required, it is suggested that interactive compositions, such as antigens or antibodies, can be immobilized onto the particles by providing active linking or binding sites on the particles to which such interactive compositions can be covalently bonded.
Another of such devices is described in U.S. Pat. No. 4,446,232 which is based on the principle of competition between bound and free species of analyte for a fixed number of recognition sites on an enzyme-labeled antibody. The determination of analyte in a test sample depends upon the binding of the analyte to enzyme-labeled antibodies in one zone of the device and which then pass into another zone of the device where the enzyme activity of the enzyme-linked antibodies bound to analyte is detected. One of the zones further includes bound and immobilized analyte which competes with analyte from the test sample for binding to the enzyme-labeled antibodies and which bind and immobilize any of the enzyme-labeled antibodies which do not become bound to analyte from the test sample.
A particular disadvantage, however, of such devices is that reverse fluid migration results in reaction products, which have migrated into the lower or detection layer, to migrate back up into the upper layers, resulting in chemical interferences and diminished test response. To overcome this disadvantage, analytical test devices have been proposed which attempt to localize or otherwise prevent such reverse fluid migration of the reaction products.
For example, European Patent Publication Nos. 51,183 and 66,648 suggest layers for collection of the detectable reaction product comprising hydrophilic high molecular weight substances. EP No. 66,648 further suggests the incorporation of mordanting agents in the detection layer which have a strong interaction with the detectable reaction product in order to collect the detectable reaction product therein. Such mordanting agents include cationic polymers, anionic polymers and quaternary salts.
Similarly, U.S. Pat. Nos. 4,144,306 and 4,042,335 disclose multilayer analytical elements which include a registration layer incorporated with a mordant for a detectable species in order to collect the detectable species therein and thereby prevent diffusion or migration of the detectable species out of the registration layer.
A variation of such devices is disclosed by U.S. Pat. No. 4,459,358 which describes a multilayer element comprising a spreading layer, a reaction layer incorporated with a diffusible labeled antibody, and a registration layer incorporated with materials adapted to non-specifically bind, immobilize or "mordant" antibodies, such as latex particles. Upon application of a liquid test medium to the device, analyte from the test medium associates with the labeled antibody in the reaction layer and immunoprecipitates therein. Any of the labeled antibody which does not become bound to the analyte diffuses into the registration layer where it is immobilized by the mordant incorporated therein.
However, the use of mordanting agents can interfere with the prerequisite reactions which are necessary for the formation or release of the detectable reaction product as a result of non-specific binding of the mordanting agent. Such interference can make both detection and measurement unreliable, as well as decrease the sensitivity of the test device.
In attempts to overcome the disadvantages of mordanting agents in a registration layer, other analytical elements have been proposed employing mordanting agents in a layer other than a registration layer in order to prevent the migration of a formed detectable reaction product into a layer other than a registration or detection layer which would otherwise render the detectable reaction product undetectable. Such a device is disclosed by U.S. Pat. No. 4,166,093 which includes a species migration-inhibiting layer interposed between a radiation-blocking layer and a reagent layer of a multilayer analytical element. The detectable species migration-inhibiting layer is permeable to analyte and fixes or otherwise prevents a significant portion of any detectable species, such as a dye formed in the reagent layer, from further migrating up into the radiation-blocking layer. Such detectable species migration-inhibiting layer comprises a mordant for the particular detectable species formed in the reagent layer. However, such an inhibiting layer still presents the disadvantage of a mordanting agent which may interfere with reactions initiated by the presence of analyte and prevent or substantially inhibit the formation or release of the detectable species.
Still another attempt to overcome the problem of reverse fluid migration in multilayer analytical elements is disclosed by International Publication No. WO 84/02193 which provides for a chromogenic support immunoassay which comprises collection of an immune complex comprising analyte bound to an enzyme-labeled anti-analyt antibody on a porous or microporous support material. The support functions to concentrate the chromatic signal generated by the label component upon reaction with signal generating reagents in the support material. Concentration of the chromatic signal results from covalent attachment of the reaction product to the support, and the problem of reverse fluid migration being overcome by providing a single layer. The immunoassay, however, requires a number of incubation and washing steps in order to localize and concentrate the signal on the support. Although the immunoassay overcomes reverse fluid migration by providing a single layer support within which the necessary reactions for production of the chromatic signal occur, it still presents the disadvantages of extensive incubation and washing steps which are not necessary with a multilayer analytical element.
Accordingly, it is an object of the present invention to overcome the aforementioned disadvantages by providing a specific binding assay in a multizone, or multilayer, test device which concentrates the detectable response of a labeled reagent without interfering with the specific binding reactions involved in the assay.
Another object of the present invention is to provide, in a multizone, or multilayer, test device, a specific binding assay having an end point in the assay where further migration of the detectable species does not occur.
Further, it is an object of the present invention to provide a sensitive specific binding assay for the highly accurate determination of analyte from a liquid test medium and which has substantially little or no background signal.